Transkingdom Network Analysis (TkNA): a systems framework for inferring causal factors underlying host-microbiota and other multi-omic interactions.

We present Transkingdom Network Analysis (TkNA), a unique causal-inference analytical framework that offers a holistic view of biological systems by integrating data from multiple cohorts and diverse omics types. TkNA helps to decipher key players and mechanisms governing host-microbiota (or any multi-omic data) interactions in specific conditions or diseases. TkNA reconstructs a network that represents a statistical model capturing the complex relationships between different omics in the biological system. It identifies robust and reproducible patterns of fold change direction and correlation sign across several cohorts to select differential features and their per-group correlations. The framework then uses causality-sensitive metrics, statistical thresholds and topological criteria to determine the final edges forming the transkingdom network. With the subsequent network's topological features, TkNA identifies nodes controlling a given subnetwork or governing communication between kingdoms and/or subnetworks. The computational time for the millions of correlations necessary for network reconstruction in TkNA typically takes only a few minutes, varying with the study design. Unlike most other multi-omics approaches that find only associations, TkNA focuses on establishing causality while accounting for the complex structure of multi-omic data. It achieves this without requiring huge sample sizes. Moreover, the TkNA protocol is user friendly, requiring minimal installation and basic familiarity with Unix. Researchers can access the TkNA software at https://github.com/CAnBioNet/TkNA/ .

Hookah Smoke Mediates Cancer-Associated Epigenomic and Transcriptomic Signatures in Human Respiratory Epithelial Cells.

INTRODUCTION: Although communal smoking of hookah by means of water pipes is perceived to be a safe alternative to cigarette smoking, the effects of hookah smoke in respiratory epithelia have not been well characterized. This study evaluated epigenomic and transcriptomic effects of hookah smoke relative to cigarette smoke in human respiratory epithelial cells. METHODS: Primary normal human small airway epithelial cells from three donors and cdk4 and hTERT-immortalized small airway epithelial cells and human bronchial epithelial cells were cultured for 5 days in normal media with or without cigarette smoke condensates (CSCs) or water pipe condensates (WPCs). Cell count, immunoblot, RNA sequencing, quantitative real-time reverse-transcriptase polymerase chain reaction, methylation-specific polymerase chain reaction, and quantitative chromatin immunoprecipitation techniques were used to compare effects of hookah and cigarette smoke on cell proliferation, global histone marks, gene expression, and promoter-related chromatin structure. RESULTS: CSC and WPC decreased global H4K16ac and H4K20me3 histone marks and mediated distinct and overlapping cancer-associated transcriptome signatures and pathway modulations that were cell line dependent and stratified across lung cancer cells in a histology-specific manner. Epiregulin encoding a master regulator of EGFR signaling that is overexpressed in lung cancers was up-regulated, whereas FILIP1L and ABI3BP encoding mediators of senescence that are repressed in lung cancers were down-regulated by CSC and WPC. Induction of epiregulin and repression of FILIP1L and ABI3BP by these condensates coincided with unique epigenetic alterations within the respective promoters. CONCLUSIONS: These findings support translational studies to ascertain if hookah-mediated epigenomic and transcriptomic alterations in cultured respiratory epithelia are detectable and clinically relevant in hookah smokers.

Retrovirus Integration Database (RID): a public database for retroviral insertion sites into host genomes.

UNLABELLED: The NCI Retrovirus Integration Database is a MySql-based relational database created for storing and retrieving comprehensive information about retroviral integration sites, primarily, but not exclusively, HIV-1. The database is accessible to the public for submission or extraction of data originating from experiments aimed at collecting information related to retroviral integration sites including: the site of integration into the host genome, the virus family and subtype, the origin of the sample, gene exons/introns associated with integration, and proviral orientation. Information about the references from which the data were collected is also stored in the database. Tools are built into the website that can be used to map the integration sites to UCSC genome browser, to plot the integration site patterns on a chromosome, and to display provirus LTRs in their inserted genome sequence. The website is robust, user friendly, and allows users to query the database and analyze the data dynamically. AVAILABILITY: https://rid.ncifcrf.gov ; or http://home.ncifcrf.gov/hivdrp/resources.htm .

Cigarette smoke mediates epigenetic repression of miR-487b during pulmonary carcinogenesis.

MicroRNAs are critical mediators of stem cell pluripotency, differentiation, and malignancy. Limited information exists regarding microRNA alterations that facilitate initiation and progression of human lung cancers. In this study, array techniques were used to evaluate microRNA expression in normal human respiratory epithelia and lung cancer cells cultured in the presence or absence of cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly repressed miR-487b. Subsequent experiments demonstrated that miR-487b directly targeted SUZ12, BMI1, WNT5A, MYC, and KRAS. Repression of miR-487b correlated with overexpression of these targets in primary lung cancers and coincided with DNA methylation, de novo nucleosome occupancy, and decreased H2AZ and TCF1 levels within the miR-487b genomic locus. Deoxy-azacytidine derepressed miR-487b and attenuated CSC-mediated silencing of miR-487b. Constitutive expression of miR-487b abrogated Wnt signaling, inhibited in vitro proliferation and invasion of lung cancer cells mediated by CSC or overexpression of miR-487b targets, and decreased growth and metastatic potential of lung cancer cells in vivo. Collectively, these findings indicate that miR-487b is a tumor suppressor microRNA silenced by epigenetic mechanisms during tobacco-induced pulmonary carcinogenesis and suggest that DNA demethylating agents may be useful for activating miR-487b for lung cancer therapy.

Lsh, chromatin remodeling family member, modulates genome-wide cytosine methylation patterns at nonrepeat sequences.

DNA methylation is critical for normal development and plays important roles in genome organization and transcriptional regulation. Although DNA methyltransferases have been identified, the factors that establish and contribute to genome-wide methylation patterns remain elusive. Here, we report a high-resolution cytosine methylation map of the murine genome modulated by Lsh, a chromatin remodeling family member that has previously been shown to regulate CpG methylation at repetitive sequences. We provide evidence that Lsh also controls genome-wide cytosine methylation at nonrepeat sequences and relate those changes to alterations in H4K4me3 modification and gene expression. Deletion of Lsh alters the allocation of cytosine methylation in chromosomal regions of 50 kb to 2 Mb and, in addition, leads to changes in the methylation profile at the 5' end of genes. Furthermore, we demonstrate that loss of Lsh promotes--as well as prevents--cytosine methylation. Our data indicate that Lsh is an epigenetic modulator that is critical for normal distribution of cytosine methylation throughout the murine genome.

Cigarette smoke induces C/EBP-ß-mediated activation of miR-31 in normal human respiratory epithelia and lung cancer cells.

BACKGROUND: Limited information is available regarding mechanisms by which miRNAs contribute to pulmonary carcinogenesis. The present study was undertaken to examine expression and function of miRNAs induced by cigarette smoke condensate (CSC) in normal human respiratory epithelia and lung cancer cells. METHODOLOGY: Micro-array and quantitative RT-PCR (qRT-PCR) techniques were used to assess miRNA and host gene expression in cultured cells, and surgical specimens. Software-guided analysis, RNA cross-link immunoprecipitation (CLIP), 3' UTR luciferase reporter assays, qRT-PCR, focused super-arrays and western blot techniques were used to identify and confirm targets of miR-31. Chromatin immunoprecipitation (ChIP) techniques were used to evaluate histone marks and transcription factors within the LOC554202 promoter. Cell count and xenograft experiments were used to assess effects of miR-31 on proliferation and tumorigenicity of lung cancer cells. RESULTS: CSC significantly increased miR-31 expression and activated LOC554202 in normal respiratory epithelia and lung cancer cells; miR-31 and LOC554202 expression persisted following discontinuation of CSC exposure. miR-31 and LOC554202 expression levels were significantly elevated in lung cancer specimens relative to adjacent normal lung tissues. CLIP and reporter assays demonstrated direct interaction of miR-31 with Dickkopf-1 (Dkk-1) and DACT-3. Over-expression of miR-31 markedly diminished Dkk-1 and DACT3 expression levels in normal respiratory epithelia and lung cancer cells. Knock-down of miR-31 increased Dkk-1 and DACT3 levels, and abrogated CSC-mediated decreases in Dkk-1 and DACT-3 expression. Furthermore, over-expression of miR-31 diminished SFRP1, SFRP4, and WIF-1, and increased Wnt-5a expression. CSC increased H3K4Me3, H3K9/14Ac and C/EBP-ß levels within the LOC554202 promoter. Knock-down of C/EBP-ß abrogated CSC-mediated activation of LOC554202. Over-expression of miR-31 significantly enhanced proliferation and tumorigenicity of lung cancer cells; knock-down of miR-31 inhibited growth of these cells. CONCLUSIONS: Cigarette smoke induces expression of miR-31 targeting several antagonists of cancer stem cell signaling in normal respiratory epithelia and lung cancer cells. miR-31 functions as an oncomir during human pulmonary carcinogenesis.